Preparation and usage of plasmodium fusion antigen

ABSTRACT

The invention provides a fusion protein comprising the  Plasmodium  merozoite surface protein-1 (MSP1) and the  Plasmodium  apical membrane antigen 1 (AMA-1), the encoding DNA sequence, the vector containing the sequence, the host cell containing the vector, and the genetic engineering method for preparing the fusion protein and the usage for producing anti-malarial vaccine. The AMA-1/MSP1 fusion protein of the present invention has excellent immunogenicity and can cause an effective immune response against  Plasmodium  in individuals.

FIELD OF INVENTION

The present invention relates to DNA recombinant techniques and geneengineering vaccines. More specifically, the present invention relatesto a fusion protein containing Merozoite Surface Protein 1 (MSP1) andApical Membrane Antigen-1 (AMA-1) of Plasmodium, a DNA sequence encodingthe fusion protein, a vector containing the DNA sequence, host cellscontaining the vector, a genetic engineering method to prepare thefusion protein, and uses of the fusion protein in developing vaccineagainst malaria.

BACKGROUND

Malaria is one of the most ancient infectious diseases that still hasstrong impact upon human health. According to the World HealthOrganization (WHO), about 40% of the world's population are still underthe threat of malaria, which has been distributed to more than 100countries. Today, there are about 300–500 million malaria cases everyyear, of which about 3 million die from it. Moreover, because of theemergence and quick spread diffusion of drug resistance by plasmodia andmosquito vector, malaria has not been effectively controlled. On thecontrary, it is likely to stage a comeback. Thus the expansion of aglobal project to control malaria has become one of the emphatic studyfields of WHO in the new century.

People can depend upon or expect to achieve breakthrough in controllingmalaria through three main pathways: anti-malaria drugs, malariavaccines and mosquito vector control. However, anti-malaria drugs andmosquito vector control are facing great difficulties and challenges,which have been the result of the emergence and spread of drugresistance of plasmodia and mosquito. Although the malaria vaccine withapplication value has not become visible at present, it is stillgenerally believed that the development of malaria vaccine is animportant pathway for human beings to control and even eradicatemalaria.

Many studies have indicated that the prevention of malaria could beachieved through the development of effective vaccines. For example,volunteers immunized by inactivated sporozoit could be completelyprotected against subsequent challenge of Plasmodium falciparum. Also,the mouse immunized by murine Plasmodium recombinant antigen could becompletely protected against subsequent infection attacks fromhomogenous Plasmodium. In addition, epidemiological studies on malariaalso demonstrates that the population dying from malaria is mainly thosehaving no immunity against malaria, e.g. children living in malariaendemic areas and population from malaria non-endemic areas enteringmalaria endemic areas, while adults in malaria endemic areas seldom dieof it. If inoculating these people having no specific immunity witheffective vaccines, it could be expected that the specific immunitycould be induced similar to that of the adults from malaria endemicareas, and thus the objective to prevent malaria and reduce malariamortality can be achieved. Hence, the development of malaria vaccineshas become the hot topic in the world nowadays.

In the development of malaria vaccines, the studies upon two candidatevaccines have ever been widely noticed, and one of which isanti-sporozoit vaccine. This vaccine could induce immunity to inhibitinvasion of sporozoit into hepatic cells. However, subsequent clinicaltrials do not show good efficacy. According to the analysis, the vaccinecould just generate immunity against sporozoit, but even if only a fewsporozoit escapes the immune attack of the vaccine and survive, theycould invade hepatic cells and develop and proliferate to generatethousands of merozoites enough to cause disease in hosts. The other oneis SPf66 polyvalence synthetic peptide vaccine. This vaccine is a45-peptide polymer made of three 10-peptide epitopes separately linkedthrough hydrophobic amino acids (Patarroyo, M. E., et al., Induction ofprotective immunity against experimental infection with malaria SuingSynthetic peptides, Nature; 328: 629, 1987). This vaccine achieved goodimmune protection against challenge of Plasmodium falciparum in Aotusmonkeys. However, an ideal clinical protection has not been achieved inthe subsequent clinical trials in Africa, South-east Asia and LatinAmerica, and thus no applicable value was established. The reason offailure of this vaccine may lie in the fact that SPf 66 only has 45peptides. In such a short, chained peptide sequence, there may not be Tcell epitopes enough for most individual MHC molecules to bind, whichresults in the prevention of antigen presentation.

Although the studies on malaria vaccines using biotechnology have beenconducted for about 20 years, a vaccine with application value has yetto become available. It can be seen that the breakthrough in malariavaccines still calls for further confirmation on the candidate antigensproviding protection against malaria, clarification on the immunologicalmechanism of protection effect, and establishment of new strategiesabout malaria vaccine development.

Thus, it is urgently required in the art to develop effective immunogensand related vaccines against malaria.

SUMMARY OF INVENTION

The purpose of the present invention is to provide a fusion proteinproduced by genetic engineering technique. The fusion protein cantrigger immune response as an effective immunogen against malaria, andthus enable the immunized individual to obtain immunity against malaria.

Another purpose of the present invention is to provide a new immunogenthat could be used in malaria vaccines. Said immunogen is a fusionprotein that contains AMA-1 and MSP1 antigens (“AMA-1/MSP1 fusionprotein”), and also to provide a vaccine composition comprising theimmunogen.

Another purpose of the present invention is to provide a DNA encodingthe fusion protein, a vector containing the DNA sequence, and host cellscontaining the vector.

Another purpose of the present invention is to provide a low-cost and/orsimple method for producing the AMA-1/MSP1 fusion protein.

The first aspect of the present invention is to provide a fusionprotein, which comprises the amino acid sequence of Plasmodium apicalmembrane antigen-1, the amino acid sequence of Plasmodium merozoitesurface protein 1, and a hinge between the amino acid sequence of theapical membrane antigen-1 and the amino acid sequence of the merozoitesurface protein 1.

More preferably; the fusion protein comprises the amino acid sequenceshown in SEQ ID NO: 1, 2 or 3.

The second aspect of the present invention is to provide an isolated DNAmolecule, which encodes the above fusion protein of the presentinvention.

The third aspect of the present invention is to provide a vector, whichcontains the DNA molecule described above, and a host cell containingthe vector.

The fourth aspect of the present invention is to provide a method forproducing the fusion protein of the present invention, which comprisesthe following steps:

growing the host cells described above under conditions appropriate forthe expression of the fusion protein, thereby expressing the fusionprotein; and

isolating the fusion protein.

The fifth aspect of the present invention is to provide a vaccine, whichcontains the fusion protein or the encoding DNA molecule described inthe present invention.

The sixth aspect of the present invention is to provide an antibody,which specifically binds to the fusion protein of the present invention.

The seventh aspect of the present invention is to provide a method forconstructing an anti-malaria multivalent vaccine, which comprises thesteps of fusing several conformational Plasmodium antigens or functionaldomains into a fusion protein molecule. In particular, the methodcomprises the following steps: (1) fusing several Plasmodium antigens orthe functional domain thereof (especially the antigen or functionaldomain with conformation) into a fusion protein molecule (in which ahinge is inserted between adjacent antigens or functional domains), andthereby obtain its amino acid sequence; (2) according to the aminosequence, designing its nucleotide encoding sequence; (3) synthesizingthe nucleotide encoding sequence (and optionally modifying the encodingsequence according to codon preference, etc.); (4) introducing thenucleotide encoding sequence into host cells to obtain the transformedhost cells; (5) incubating the transformed host cells under properconditions to express the fusion protein; (6) isolating or purifying thefusion protein to use as an anti-malaria multivalent vaccine.

DESCRIPTION OF DRAWINGS

FIG. 1: Schematic diagrams of PfCP-1 (FIG. 1A) and PfCP-2 (FIG. 1B)fusion protein and the N and C terminal sequences thereof. Ectodomain ofAMA-1 was suggested to be divided into three regions, namely regions I,II and III. MSP1-19 is the 19KD C terminal region of MSP1. MCS is amultiple cloning site region that contains 8 single restriction cleavagesites, which is used for further insertion of other antigen genes. H isa hinge sequence, which is made of repeated sequences of Gly-Pro-Gly,and is used to prevent the two proteins from interacting in conformationwith each other.

FIG. 2 is the synthetic strategy schematic diagram for the fusionantigen gene PfCP-1 (FIG. 2A) and PfCP-2 (FIG. 2B).

FIG. 2A: Synthesis of PfCP-1 gene: 1997 bp PfCP-1 gene is divided into 4segments and each of the segments is synthesized separately. The nameand length of these 4 segments are: PfCP-1a, 512 bp; PfCP-1b, 629 bp;PfCP-1c, 575 bp; PfCP-1d, 395 bp respectively. XhoI and EcoRI sites areadded to the 5′ and 3′ end of each segment for gene segment cloning.Each synthesized segment is first cloned into a pBluscript vector andthen undergoes sequence analysis. Each segment is linked to generate thefull-length PfCP-1 gene through ScaI, HindIII and KpnI sites shown inthe figure.

FIG. 2B: Synthesis of PfCP-2 gene: a pair of primers are designedrespectively at the start point (Pa) of PfCP-1 AMA-1 (III) and the 3′terminal (Pb), which is obtained through amplification with PCR method.

FIG. 3: Agarose gel electrophoresis showing the full-length PfCP-1 gene.After cutting with XhoI and EcoRI enzyme, electrophoresis shows thePfCP-1 synthesized gene and the vector bands (lane 1).

FIG. 4: the schematic diagram for the pPIC9k/PfCP-1 recombinantexpression plasmid.

FIG. 5: the schematic diagram for the pPIC9k/PfCP-2 recombinantexpression plasmid.

FIG. 6: Western blot detection of PfCP-1 expression, in which lane 2 isthe supernatant from the culture before induction, and lane 1 is thesupernatant from the culture at 72 hours after induction. The monoclonalantibody mAb5.2 which recognizes the conformation epitopes of MSP1-19and the secondary antibody anti-mouse IgG are used for detection.

FIG. 7: the detection of PfCP-2 expression by SDS-PAGE. 10 ul of culturesupernatant is loaded on the SDS-PAGE gel and Coomassie-stained at 0 hrbefore induction and 24, 48, 72 and 96 hr after induction. The doubletof the products are presented at the 32KD location. N-terminal aminoacid sequence analysis of the doublet shows that 9 amino acid residuesare deleted at the N-terminal of the lower band.

FIG. 8: the reaction of a monoclonal antibodies with PfCP-2. The mAbsZF10, IE1, 2.2, 111.2, 12.8 and 5.2 are all specific monoclonalantibodies used to identify conformation epitopes. The mAb 9.8 is thenegative control, and PCAB is anti-AMA-1 polyclonal antibody. “−” meansthat β-mercapto-ethanol was not present in the sample; “+” means thatβ-mercaptoethanol was added in the sample. The illustration involves theprocedures of applying equal amount of PfCP-2 expression supernatant toeach lane, performing electrophoresis and transferring to the membrane,and using the above antibodies and the corresponding secondary antibodyto proceed with reaction and staining.

FIG. 9: the SDS-PAGE gel showing the purified PfCP-2 recombinantprotein. The expression product was purified from the supernatant in twosteps with Ni column and gel filtration chromatography. According toSDS-PAGE and HPLC measurement, the purity of the target protein is above98%. Each lane respectively is: lane 1, 10 ug; lane 2, 20 ug; and lane3, 30 ug.

FIG. 10: the PfCP-2 specific IgG level of the immunized rabbit serummeasured by ELISA. Each group respectively is: ISA720 adjuvant+PfCP-2;ISA720 adjuvant+denatured PfCP-2; Freund's adjuvant; and Freund'sadjuvant+PfCP-2.

FIG. 11: the PfCP-2 specific IgG level of the immunized rabbit serummeasured by IFA. Each group respectively is: ISA720 adjuvant+PfCP-2;ISA720 adjuvant+denatured PfCP-2; Freund's adjuvant; and Freund'sadjuvant+PfCP-2.

FIG. 12: the anti-AMA-1 and MSP1 specific IgG level of the immunizedrabbit serum measured by ELISA. AMA-1 expressed and refolded in E. coliand MSP1-19 expressed in yeast are respectively used as the antigens tomeasure each specific antibody, in which the horizontal scale is thedifferent doses of the immune serum of the PfCP-2 antigen immunizedrabbit; and the ordinate scale is the antibody titer measured by ELISA.

FIG. 13: the in vitro inhibition of the parasite growth by immunesera(1). Each group respectively is: 1. Freund's adjuvant+PfCP-2 (15%serum concentration), and 2. Freund's adjuvant.

FIG. 14: the in vitro inhibition of the parasite growth by immunesera(2). Each group is: 1. ISA720 adjuvant; and 2. ISA720adjuvant+PfCP-2.

FIG. 15: the in vitro inhibition of the parasite growth by immunesera(3). Each group is: 1. ISA720 adjuvant+PfCP-2; and 2. ISA720adjuvant+denatured PfCP-2.

FIG. 16: the in vitro inhibition of the parasite growth by immunesera(4). Each group is: 1. ISA720 adjuvant; 2. ISA720 adjuvant+PfCP-2;3. ISA720 adjuvant+denatured PfCP-2; 4. Freund's adjuvant+PfCP-2; and 5.Freund's adjuvant.

DETAILED DESCRIPTION

The studies have indicated that the life cycle of plasmodia iscomplicated, and the antigens stage-specific. Besides, there is severeantigen variation in Plasmodium. Based upon these characteristics, aneffective and continuously applicable malaria vaccine should includequite a few Plasmodium protective antigens, from which the immunitygenerated should be capable of attacking different stages of theparasite. Thus, even if a few parasites could survive under the immuneattack from the first defensive line for reasons such as antigenmutation, these parasites still will experience attacks from eachsubsequent defensive line, until all the parasites are eliminated. Atpresent, more than 20 proteins have been considered as the candidateantigens for malaria vaccine. Seven of the candidate antigens were mixedto form a multivalent vaccine, and clinical trial was performed. Among35 volunteers receiving vaccination, only 1 had obtained protection. Asmeasured, the immunized individuals have very low antibody titer foreach antigen. This may be caused by the antigen competition when variousantigens injected simultaneously, and thus affect the immune response ofthe body to each antigen.

Another practical problem about malaria vaccines is human MHCpolymorphism. Because of the polymorphism, some antigens can not bind toMHC molecules, and it leads to the inhibition of antigen presentation,and the non immune responses phenomenon in immunized individuals. Toensure that the vaccine would be effectively presented in mostindividuals, it is necessary to identify the multi-reaction epitopesfrom malaria antigens that could recognize MHC molecules in mostpopulation, and incorporating them into a vaccine, or incorporatingvarious antigens to form a fusion antigen to overcome the problem withlack of enough T cell epitopes in unique antigen are required.

An effective malaria vaccine needs to incorporate multiple antigens,while the simple mixture of multiple antigens may cause the problem ofantigen competition. Therefore, we developed another route to constructthe multivalent vaccine antigen after wide and deep research, i.e. firstidentify the protective domains or regions from existing candidateantigens. These functional domains were then assembled to a fusionprotein via appropriate design, and the gene of the protein wasredesigned and synthesized. finally, the recombinant protein wereexpressed. Through the designing and insertion of a hinge sequence, thefunctional domains in this fusion protein could maintain theirconformation. As a result, it is found that the fusion comprisingmerozoite surface protein 1 (MSP1) and apical membrane antigen-1 (AMA-1)of Plasmodium falciparum could effectively induce protective immuneresponse. And the invention is accomplished upon this basis.

The fusion protein of the present invention involves two antigens ofPlasmodium falciparum. One is merozoite surface protein 1 (MSP1) and theother is apical membrane antigen-1 (AMA-1). Both of them are importantcandidate antigens for malaria vaccines at present. The Aotus monkeysimmunized with the MSP1 extracted from cultured Plasmodium falciparumcould be completely protected against challenge of homologous strainPlasmodium falciparum (Siddqui, W. A. et al., Merozoite surface coatprecursor protein completely protects Aotus monkeys against Plasmodiumfalciparum malaria. Proc. Natl. Acad. Sci. USA, 84: 3014, 1987). Manyexperimental evidences have indicated that MSP1 19 KD C-terminal segment(MSP1-19) is the functional domain for protective immunity of thisantigen. Both MSP1-19 immune serum and monoclonal antibody could inhibitthe in vitro growth of malaria parasite. This domain contains 10cysteine residues, which form two Epidermal Growth Factor (EGF) likedomains. The Aotus monkeys immunized with recombinant protein containingMSP1-19 could also be protected against subsequent challenge.

AMA-1 is a membrane protein with about 60 KD molecular weight. Immuneserum against the ectodomain inhibited the in vitro growth of theparasite. The mouse immunized with an analogous of mouse PlasmodiumAMA-1 could also be protected against challenge of the same species ofthe Plasmodium. AMA-1 contains 16 conserved cysteine residues, forming 3domains linked by disulfide bonds, wherein domain III[AMA-1 (III)] isvery conserved in sequence. It is presumed that the newly invadingmerozoite still carries this domain sequence, which may take part in themerozoite invasion.

The result of studies indicates that the protective effects of bothAMA-1 and MSP1-19 are dependent upon the conformation formed bydisulfide bonds in the antigens. Reduced and alkylated antigens failedto generate an effective immune protection effect. Hence, the key infusing these two antigens into one molecule is to maintain the naturalconformation of each antigen.

The present invention has constructed two fusion antigens AMA-1/MSP1-19(named as PfCP-1) and AMA-1 (III)/MSP1-19 (named as PfCP-2), andexpressed these antigens in yeast Pichia pastoris. The analysis showsthat the protein resembled very close to the natural one. This antigenis highly immunogenic with ELISA titer at >4 million. The antibodies ofthe fusion antigen recognized the individual AMA-1 and MSP1 protein ofthe fusion protein. The immune serum against the fusion antigencompletely inhibited the growth of the parasite in vitro after beingdiluted 6.7 times.

As used herein, the terms “the fusion protein of merozoite ApicalMembrane Antigen-1 and Merozoite Surface Protein 1”, and “AMA-1/MSP1fusion protein” are used interchangeably, and both mean the fusionprotein comprising the amino acid sequence of Plasmodium MerozoiteSurface Protein 1, and the amino acid sequence of Plasmodium merozoiteApical Membrane Antigen-1, between which there may or may not be hingesequence. In addition, the fusion protein of merozoite Apical MembraneAntigen-1 and Merozoite Surface Protein 1 may or may not contain asignal peptide, and may or may not contain an initial methionine.

As used herein, the term “Plasmodium merozoite Apical Membrane Antigen-1(AMA-1) amino acid sequence” refers to a part of the amino acid sequenceof the fusion protein of the present invention. The sequence basicallyhas the same amino acid sequence with natural sequence and segmentthereof, and basically has the same antigen activity with naturalPlasmodium AMA1. A preferred Plasmodium AMA-1 amino acid sequencecomprises (but is not limited to): the amino acid sequence of naturalAMA-1, the amino acid sequence of the whole ectodomain of AMA-1, theamino acid sequence of domain III of AMA-1, the amino acid sequence ofdomain I-III of AMA-1, and the amino acid sequence with glycosylationsites eliminated.

As used herein, the term “Merozoite Surface Protein 1 (MSP1) amino acidsequence” refers to a part of the amino acid sequence in the fusionprotein. This sequence basically has the same amino acid sequence withnatural MSP1 sequence of Plasmodium, and basically has the same antigenactivity with natural MSP1. A preferred Plasmodium MSP1 amino acidsequence comprises (but is not limited to): the amino acid sequence ofnatural MSP1, the amino acid sequence of MSP1 19 KD C-terminal, and theamino acid sequence with glycosylation sites eliminated.

The amino acid sequences of AMA-1 and MSP1 and analogues thereof couldbe obtained from different Plasmodium such as human Plasmodium (forexample, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae,and Plasmodium ovale), and animal Plasmodium (such as mouse Plasmodium,monkey Plasmodium, etc.).

There is no limitation on the linking mode or order between the AMA-1amino acid sequence and the MSP1 amino acid sequence. E.g, it can behead-tail linking, head-head linking or tail-tail linking.

As used herein, the term “hinge” refers to a short peptide between theAMA-1 amino acid sequence and the MSP1 amino acid sequence used inlinking them. There is no special limitation on the length of the hinge.It may even be 0 when the AMA-1 amino acid sequence directly link withthe MSP1 amino acid sequence. Usually, the hinge will not significantlyaffect the formation of correct folding and special conformation of theAMA-1 amino acid sequence and the MSP1 amino acid sequence. Someexamples of the hinge include (but is not limited to):

Preferably, the hinge has the following amino acid sequence:

(a) An amino acid sequence containing 3–15 amino acids made ofhydrophobic amino acids Gly and Pro. For example Gly-Pro-Gly-Pro-Gly-Pro(SEQ ID NO: 7);

(b) An amino acid sequence encoded by Multiple Cloning Sites. Thissequence usually contains 5–20 amino acids, and preferably 10–20 aminoacids. The example comprises (but is not limited to): TGLQPTRGIDDITSPVD(SEQ ID NO: 8);

(c) A Plasmodium antigen amino acid sequence except AMA-1 and MSP1, suchas Plasmodium Circumsporozoit Protein, TRAP, and 175KD ErythrocyticBinding Protein, etc.

(d) A combined amino acid sequence of (a), (b) and/or (c). One exampleof the hinge made by (a) and (b) is GPGPGTGLQPTRGIDDITSPVDGPGPGP (SEQ IDNO: 9).

In addition, an amino acid sequence that does not affect theimmunogenicity of AMA-1 and MSP1 could be added to the N-terminal orC-terminal of the AMA-1/MSP1 fusion protein. Preferably, these addedamino acid sequences could help expression (such as a signal peptide),purification (such as 6His tag, alpha-factor signal peptide cleavagesite (Glu-Lys-Arg) in Saccharomyces cerevisiae), or could enhance theimmunogenicity of an AMA-1/MSP1 fusion protein (for example, thesequence of cytokine, such as interferon and IL, etc.).

The total DNA sequence encoding the fusion protein of the presentinvention could be artificially synthesized. The encoding DNA sequenceof AMA-1 and/or MSP1 could also be obtained through PCR amplification orsynthesis, and then be ligated together to form the DNA sequenceencoding the fusion protein of the present invention.

To increase the expression yield in host cells, alterations could bemade to the encoding sequence of the AMA-1/MSP1 fusion protein, forexample, using the codons preferred by the host cells, or eliminatingthe sequence adverse to gene transcription and translation. In oneexample of the present invention, the codons preferred by yeast wereadopted, and the sequence in the gene adverse to gene transcription andtranslation, comprising intron cleavage site, transcription terminationsequence, etc. were eliminated. ScaI, HindIII and KpnI unique cleavagesites were incorporated at nucleotide base 494, 1085 and 1621respectively of this gene to facilitate gene synthesis and cloning.

After obtaining the DNA sequence encoding the fusion protein of thepresent invention, it was incorporated into proper expression vector,and then transferred to proper host cells. Finally, the transformed hostcells were cultured, and the fusion protein of the present invention wasobtained through expression and purification processes.

As used herein, the term “vector” includes a plasmid, a cosmid, anexpression vector, a cloning vector and a virus vector, etc.

In the present invention, various vectors known in the art such ascommercially available vectors could be used. For example, acommercially available vector can be operably linked to the nucleic acidsequence encoding the new fusion protein in the present invention underan expression regulatory sequence, and thus forms the expression vector.

As used herein, the term “operably linked” refers to such a conditionwhere a certain part of a linear DNA sequence could affect the activityof another certain part in the same linear DNA sequence. For example, ifa signal peptide plays a role in the secretion of polypeptides, then theDNA sequence encoding the signal peptide (precursor sequence ofsecretion) is operably linked to the DNA of the polypeptide; if apromoter controls the transcription of a sequence, then it is operablylinked to the encoding sequence. If a ribosomal binding site is locatedat a position which can initiate translation, then it is operably linkedto the encoding sequence. Generally, “operably linked” means adjacency,while in the precursor sequence of secretion it means adjacency inreading frame.

As used herein, the term “host cell” comprises prokaryotic cells andeukaryotic cells. The commonly used prokaryotic host cells comprise E.coli, Bacillus subtilis, etc. The commonly used eukaryotic cellscomprise yeast cells, insect cells, mammal cells, etc. Preferably, thehost cells are eukaryotic cells, more preferably yeast cells.

After obtaining the transformed host cells, the host cells could becultured under proper conditions to express the fusion protein of thepresent invention. Then the expressed fusion protein is isolated.

In another aspect, the present invention further comprises the specificantibody against the AMA-1/MSP1 fusion protein, particularly themonoclonal antibody. “Specific” herein means that the antibody can bindto the AMA-1/MSP1 fusion protein or its segment. Preferably, the termrefers to those antibodies that can bind to the AMA-1/MSP1 fusionprotein or its segment but do not recognize and bind to othernon-relative antigen molecules. The present invention further comprisesthose antibodies that can bind to the AMA-1/MSP1 fusion protein inmodified or unmodified forms.

The present invention comprises not only an intact monoclonal orpolyclonal antibody, but also an antibody segment with immune activity,such as Fab′ or (Fab)₂ segment; a heavy chain of antibody; a light chainof antibody; a single chain Fv molecule reconstructed by geneticengineering; or a chimeric antibody.

The antibodies of the present invention can be prepared through varioustechniques well known by persons skilled in the art. For example, thepurified AMA-1/MSP1 fusion protein or the segment with immunogenicitythereof can be administrated to an animal to induce the generation of apolyclonal antibody. Similarly, cells that express the AMA-1/MSP1 fusionprotein or the segment with immunogenicity thereof can be used toimmunize an animal for the generation of antibody. The monoclonalantibody of the present invention can be prepared through the hybridomatechnique (see Kohler et al., Nature 256: 495, 1975; Kohler et al., Eur.J. Immunol. 6: 511,1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976;Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas,Elsevier, N.Y., 1981).

The production of polyclonal antibody can be achieved by using theAMA-1/MSP1 fusion protein or polypeptide to immunize animals such asrabbit, mouse, rat, etc. Various adjuvants can be used to increaseimmune reaction, which comprise, but are not limited to, Freund'sadjuvant.

In another aspect of the invention, a vaccine containing the fusionprotein of the present invention is provided. The vaccines of thepresent invention are mainly prophylactic (i.e. to prevent frominfection).

These vaccines include an immune antigen or an immunogen, an immunogenicpolypeptide, protein or protein segment, or a nucleic acid (such as RNAor DNA), and are usually combined with a “pharmaceutically acceptablecarrier” which include any carrier that does not itself induce theproduction of antibodies harmful to the individual receiving thecomposition. Suitable carriers are typically large, slowly metabolizedmacromolecules such as proteins, polysaccharides, polylactic acids,polyglycolic acids, polymeric amino acids, amino acid copolymers, lipidaggregates (such as oil droplets or liposomes), and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art. Additionally, these carriers may function as immunostimulatingagents (“adjuvants”). Furthermore, the vaccine composition of thepresent invention could further contain other immunogenic Plasmodiumproteins, such as Plasmodium circumsporozoite Protein and an immunogenicsegment or a fusion protein thereof.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc., (2) ISA 720adjuvant, and (3) Freund's adjuvant, etc.

The vaccine composition (comprising an antigen, a pharmaceuticallyacceptable carrier and/or an adjuvant), usually comprises a diluent,such as water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. Furthermore,the vaccine composition, including immunogenic composition, may containthe antigen, polypeptide, protein, and protein segment or nucleotideacid together with pharmaceutically acceptable vehicles.

More specifically, vaccines comprising immunogenic compositions comprisean immunologically effective amount of the immunogenic polypeptides, aswell as any other of the above-mentioned components, as needed. By“immunologically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated (e.g., nonhumanprimate, etc.), the capacity of the individual's immune system tosynthesize antibodies, the degree of protection desired, the formulationof the vaccine, the treating doctor's assessment of the medicalsituation, and other relevant factors. It is expected that the amountwill fall in a relatively broad range that can be determined throughroutine trials.

Typically, the vaccine compositions or immunogenic compositions areprepared as injectables, either as liquid solutions or emulsions; solidforms suitable for solution in, or suspension in, liquid vehicles priorto injection may also be prepared. The preparation also may beemulsified or encapsulated in liposomes for enhanced adjuvant effect, asdiscussed above under pharmaceutically acceptable carriers.

The immunogenic compositions are conventionally administeredparenterally, e.g., by injection, either subcutaneously orintramuscularly. Additional formulations suitable for other modes ofadministration include oral formulations, suppositories, and transdermalapplications. Dosage treatment may be a single dose schedule or amultiple dose schedule. The vaccine may be administered in conjunctionwith other immunoregulatory agents.

A model of vaccine is DNA vaccine, i.e. the vaccine that contains theDNA sequence encoding the fusion protein of the present invention.

The advantages of the present invention lie in:

(1) In the AMA-1/MSP1 fusion protein, after fusion of the AMA-1 andMSP1-19 into one molecule, the conformations thereof are very close totheir respective natural conformations, with at least 6 monoclonalantibody epitopes being identical to that of the natural proteins.

(2) The expression product of the AMA-1/MSP1 fusion protein could besecreted into the protein-free culture supernatant, which will beconvenient for isolation and purification. The purity could achievehigher than 98%.

(3) The expression level of the AMA-1/MSP1 fusion protein is high.Particularly, the expression level of PfCP-2 is extremely high. Theexpression yield in a shaking flask is 840 mg/L, while the expressionyield in a 15-L ferment pot could achieve 2,600 mg/L.

(4) The AMA-1/MSP1 fusion protein is very high immunogenic. The ELISAantibody titer thereof is more than 4 million, and this antibodies alsocontain AMA-1 and MSP1 specific antibodies.

(5) The immune serum against the AMA-1/MSP1 fusion protein has verystrong inhibition upon in vitro growth of the parasite. This immuneserum could inhibit more than 98% of the parasite growth in vitro afterbeing diluted 6.7 times.

The invention is further illustrated by the following examples. Theseexamples are only intended to illustrate the invention, but not to limitthe scope of the invention. For the experimental methods in thefollowing examples, they are performed under routine conditions, e.g.,those described by Sambrook. et al., in Molecule Clone: A LaboratoryManual, New York: Cold Spring Harbor Laboratory Press, 1989, or asinstructed by the manufacturers, unless otherwise specified.

EXAMPLE 1 Synthesis of PfCP-1 and PfCP-2

1. Design of the Fusion Antigen

The amino acid sequence of the fusion protein derived from extodomain ofAMA-1 and MSP1-19 of Plasmodium falciparum 3D7 line. A hinge sequencecomprising hydrophobic amino acid Gly-Pro was inserted into the twoantigens to avoid the interaction of the antigens and retain theirnative conformation. In addition, 8 restriction sites were insertedbetween the two antigens, which allowed further insertion of otherantigens. The XhoI site and Saccharomyces cerevisiae alpha-factor signalpeptide cleavage sequence were introduced to the N-terminus of thefusion protein. Three potential glycosylation sites were eliminated bysubstituting Ala for Asn. The fusion antigen PfCP-1 comprised 660 aminoacids while PfCP-2 260 amino acids. The schematic diagram of the twoantigens and N- and C-terminal sequence of the antigens were shown inFIG. 1.

2. Design of the Synthetic Gene

The amino acid sequence of PfCP-1 was reverse-translated into DNAsequence using codon usage optimized for expression in yeast. Thedesigned sequence was examined and modified to exclude sequences whichmight cause problem during synthesis, cloning and expression of thegene. They included putative splice donor, acceptor sites and terminatorsequences, etc. The unique restriction sites ScaI, HindIII and KpnI wereintroduced for gene synthesis and cloning at position 494, 1085 and1621, respectively (FIG. 2). The entire synthetic PfCP-1 gene comprised1997 bp while PfCP-2 797 bp.

3. Synthesis of the Fusion Antigen Gene

Construction of PfCP-1 Gene

The sequences of the gene containing 1997 bp were divided into fourfragments named PfCP-1a, PfCP-1b, PfCP-1c and PfCP-1d, respectively.There was an overlapping region between two adjacent fragments and aunique restriction site was designed to ligate the fragments (FIG. 2A,indicated by arrow). Thus, unique restriction sites Sca I, Hind III andKpn I were introduced at position 494, 1085 and 1621 bp.

The 512 bp PfCP-1a fragment was synthesized with 8 oligonucleotidesusing PCR-based synthesis method. The PCR reaction was performed bydenaturation at 95° C. for 10 sec, annealing at 55° C. for 30 sec andelongation at 72° C. for 90 sec with 30 cycles. The PCR products wereanalyzed on the 1% agarose gel and purified using Qiagen DNA gelpurification Kit. The gene fragment and vector pBluescript were digestedwith XhoI and EcoRI. In 20 ul reaction system, 2 ug DNA was digested at37° C. for 1 hr with 5 units of enzyme XhoI and EcoRI, respectively. Thedigested gene fragment and vector were ligated to generate a recombinantplasmid. E. coli DH5α was transformed with the recombinant plasmid. Amp⁺transformants were selected for isolation of plasmid containing thetarget gene. DNA sequencing of target gene was carried out and theresults showed that one of three clones had error-free. Similar strategywas used to assemble the rest fragments, i.e. PfCP-1b, PfCP-1c andPfCP-1d. The fragments were recovered using appropriate restrictionenzymes XhoI/ScaI, ScaI/HindIII HindIII/KpnI and KpnI/EcoRI and ligatedinto the vector digested by XhoI/EcoRI, thereby forming gene PfCP-1which encoded a fusion protein having amino acid sequence of SEQ IDNO:1.

E. coli strain DH5α was transformed with the recombinant plasmidcontaining the entire gene. The synthetic gene is stable in E. colibecause no error on the gene recovered from the bacteria after severalpassages was observed.

To express the PfCP-1 gene in Pichia pastoris, the DNA sequence encodingLys-Arg sites was introduced to the 5′-terminus of the gene to recreatethe alpha-factor signal peptide cleavage sites. To facilitatepurification, 6× his tag was added to the 5′-terminus of the gene togenerate a PfCP-1^(+his) while PfCP-1^(−his) had no tag. Unique siteXhoI and EcoRI were introduced at the 5′-terminal and 3′-terminal of thegene, respectively to facilitate the cloning of the gene into expressionplasmid.

Construction of PfCP-2 Gene

To construct PfCP-2 gene, a pair of primers were synthesized. The 5′primer: 5′-ccg ctc gag aaa aga caa caa tca tct tac att g-3′ (SEQ ID NO:4) was corresponding to postion 1234 of PfCP-1 gene. The 3′ primer:5′-cg gaa ttc cta tta atg atg atg atg atg atg att aga gga aga gca gaag-3′ (SEQ ID NO: 5) hybridized to the C-terminal sequence. Using the twoprimers, we amplified the 797 bp PfCP-2 gene encoding amino acidsequence of SEQ ID NO:2 using PfCP-1 as template DNA which encodes theprotein containing 6× his tag.

The PfCP-2 gene encoding amino acid sequence of SEQ ID NO: 3 without6His tag was generated using another 3′ primer 5′-cg gaa ttc cta tta attaga gga aga gca gaa g-3′ (SEQ ID NO: 6).

EXAMPLE 2 Expression of PfCP in Pichia pastoris

2.1 Construction of Expression Vector

PfCP-1 or PfCP-2 was cloned in yeast expression vector pPIC9 using XhoIand EcoRI sites. The Saccharomyces cerevisiae alpha-factor signalpeptide and its cleavage site (Lys-Arg) was introduced in frame with thePfCP coding sequences. Thus the mature secretary proteins should notcontain any signal peptide residue.

The vector pPIC9K was identical to pPIC9 except for the presence of theKanamycin resistance gene to screen for multiple-copy insertion. Usingthe BamHl and SalI sites on the vectors, transferring the BamHl/SalIfragments from pPIC9 to pPIC9k to generate pPIC9K/PfCP-1 (FIG. 4) andpPIC9K/PfCP-2 vectors (FIG. 5).

2.2 Transformation of Pichia pastoris SMD1168

The pPIC9K/PfCP-1 and pPIC9K/PfCP-2 was linearized with SalI.Transformation of Pichia pastoris strain SMD1168 was carried out byelectroporation with 10 ug of linearized DNA. The His⁺ transformantswere further screened for transformation with multiple-copy insertion onYPD plates containing various concentration of G418. The insertion oftarget gene need to be confirmed by PCR using genomic DNA isolated fromthe His+/G418 positive clones. The clones confirmed by PCR were used forexpression.

The Pichia pastoris strain expressing PfCP-1 was deposited at the ChinaCenter for Type Culture Collection (CCTCC) on Nov. 22, 2000 with theaccesion number of CCTCC NO:M 200026.

2.3 Expression

The transformants were grown in MGY medium containing glycerol for 24hrs. The cells were harvested and the pellet was resuspended in BMMYmedium containing methanol to induce expression. The supernatant as wellas cell pellet were analyzed for protein expression by coomassiestrained SDS-PAGE gel every 24 hr. The result showed that after methanolinduction, a PfCP-2 band appeared at 32 kD. The expression productsignificantly increased with the elongation of time (FIG. 7). Specificmonoclonal antibody mAb5.2 was used to detect the product and the resultshowed that the dimmer and tetramer of PfCP-2 was detected besides the32 kD protein (FIG. 7B). In addition, the doublet of PfCP-2 protein wasobserved on the SDS-PAGE gel. N-terminal sequence analysis of theproduct showed that the doublet had different N-terminus, with 9 aminoacid deletion in the band with low molecular weight. Expression ofPfCP-1 protein was detectable with a band at 72 kD by Western blot butvery week band appeared on SDS-PAGE and Coomassie stained gel (FIG. 6).

EXAMPLE 3 Interaction of the AMA-1/MSP-1 Fusion Protein and a Panel ofMonoclonal Antibodies

MSP1-19 and AMA-1 are cysteine-rich proteins that form several disulfidebonds. Moreover, their functional antibodies are disulfidebond-dependent. Thus the key issue for this invention is to retain allconformational epitopes after fusion of the two proteins into onemolecule. Therefore, to gain an insight into conformational propertiesof the fusion proteins, in this experiment a panel of monoclonalantibodies were used to react with PfCP-1 and PfCP-2 fusion protein.

Total 13 monoclonal antibodies, of them 10 recognizing conformationalepitopes, were used to interact with PfCP-1. The expression productswere subjected to electrophoresis in SDS-PAGE under non-reducingconditions, and then transferred onto pyroxylin membrane. Themembrane-bound proteins were reacted with each of the 13 mAbsrespectively and visualized via the AP-conjugate secondary antibodyfollowing standard procedures. The results showed that all 13 specificmAbs interacted with the expression protein (Table 1).

TABLE 1 Interaction of PfCP-1 with a panel of monoclonal antibodiesWestern blot PfCP Specific Antibodies PfCP-1 Interaction from P. AntigenSpecificity mAb No. Epitope type region pastoris MSP-1 Conserved 5.2conformational MSP 1–19 + 12.8 conformational MSP 1–42 + 12.10conformational MSP 1–19 + 2.2 conformational MSP 1–19 + 6.1conformational MSP 1–42 + 1E1 conformational MSP 1–19 + 2F10conformational MSP 1–19 + 8A12 conformational MSP 1–19 + 111.4conformational MSP 1–19 + 111.2 conformational MSP 1–19 + AMA-1 3D7 1F9AMA-1 + 2C5 AMA-1 + 5G8 AMA-1 +

Six conformational monoclonal antibodies were used to react with PfCP-2protein under reducing and non-reducing conditions. 6% β-mercaptoethanolwas added to the sample to reduce the protein. Same amount of reducedand non-reduced protein was loaded on each lane of SDS-PAGE gel and wasdetected with the antibodies according to the procedures describedabove. The results showed that all the monoclonal antibodies interactedwith PfCP-2 in reduction-sensitive manner (FIG. 8).

Conclusion: The fusion protein resembles closely to native conformationafter fusing of AMA-1 and MSP1-19 into one molecule. At least the sixepitopes recognized by the antibodies are identical to the native ones.

EXAMPLE 4 Fermentation and Purification of AMA-1/MSP 1 Fusion Protein

Expression conditions were optimized to achieve the yield of 840 mg/L inflask expression.

During fermentation of the yeast strain in 15-liter fermentor, the cellsgrow fast and increase by index exponent. The cell density could reachOD₆₆₀=112.5 value. During the period of methanol induction, the celldensity was maintained at the same level while the target proteinappeared in the first 3–7 hours and then dramatically increased. By 53hours after induction, the expression yield reached 2600 mg/L.

PfCP-2 expression product was purified by two steps. At the first stepprotein was purified with Ni-NTA column because 6His residues located atthe C-terminus of PfCP-2 that can combine to the Ni-NTA chelate. Thebound proteins were eluted by a buffer containing 250 mM imidazole. Atthe second step, proteins eluted from Ni-NTA were further purified bygel filtration chromatography. The protein of >98% purity was obtainedby the two-step purification (FIG. 9).

EXAMPLE 5 Immunizing Rabbit with PfCP-2

In this experiment, rabbits were immunized with the purified PfCP-2protein formulated with either Freund's adjuvant or Montanide ISA 720adjuvant, respectively. The animals were divided into 5 groups. Thegroups 1–5 were ISA 720, ISA 720+PfCP-2, ISA 720+denatured PfCP-2,Freund's+PfCP-2 and Freund's adjuvant, respectively. The rabbits wereimmunized four times on Day 0, 12, 28 and 42, respectively. Before and 7days after each immunization, sera was prepared by bleeding. Specificantibodies were analyzed. Methods involving this experiment wasdescribed as the following:

PfCP-2 antigen was formulated with Freund's adjuvant at ratio 1:1 whilewith ISA 720 adjuvant at 3:7 (v/v). The denatured PfCP-2 antigen wasprepared as following: the antigen solution was mixed with solid urea tofinal concentration of 8M and incubated at 50° C. for 60 min. DTT wasadded to a final concentration of 20 mM and incubated at 50° C. for 6hr. Sodium iodoacetate was added to a final concentration of 60 mM andmixture was incubated at room temperature (RT) for 60 minutes. Theprotein solution was dialysed in 0.1M boric acid buffer overnight (100mM boric acid, 137 mM NaCl, PH 7.5). The rabbits were inoculatedintromuscularly four times at 0, 12, 28 and 42 days. The protein dosewas 800, 400, 400 and 200 ug, respectively. The animals were bleeded onDay 10, 26, 39 and 47 and sera were analyzed for specific antibodies byELISA and IFA.(FIGS. 10,11)

The protocol for ELISA: 96-well microtiter plates were coated with 0.1ug of PfCP-2 or P. flaciparum (Pf) parasites proteins. 100 ul dilutedsera were added to each well and incubated for 1 h at 37° C. Everysample was repeated in three wells. After washing, enzyme-labelsecondary antibodies was added to each well. The incubation and washingwere carried out as described above. TMD was used as substrate. Theplates were read at an absorbance of 450 nm. Cutoff values weredetermined as the mean plus three standard deviations for thepre-immunization sera.

The ELISA results showed that no specific antibodies was induced in bothadjuvant control groups, but significant specific antibodies was inducedin the other three immunized groups. P. flaciparum parasites obtainedfrom in vitro cultivation were used as antigens for IFA. Groups 2 and 4had higher level of specific antibodies with IFA titer of 1:10240 and1:2560 respectively while the group of denatured protein (Group 3) hadmuch lower level of the antibodies with titer only 1:640. As expected,sera from the two control groups had not antibodies recognizing theparasite (<1:40) (FIG. 11). The sera of rabbits immunized with PfCP-2was further tested by ELISA for specific IgG against the individualcomponents of the fusion protein, AMA-1 and MSP1-19. The antigens usedfor this testing included the AMA-1 that was produced in E. coli andrefolded as well as MSP 1-19 that was expressed in yeast. It wasdemonstrated that rabbit sera interacted with the individual componentsof AMA-1 and MSP 1-19 specifically (FIG. 12).

EXAMPLE 6 In Vitro Growth Inhibition Assay

FCC1/HN line of P. flaciparum was cultured in vitro using Trager'sCandle Jar method. The inhibition assay was performed by using thismethod. The inhibition rate was calculated according to the followingformula:

${{Inhibit}\mspace{14mu}{rate}\;({IR})} = {\frac{\begin{matrix}{{{parasitaemia}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{group}} -} \\{{parasitaemia}\mspace{14mu}{in}\mspace{14mu}{experimental}\mspace{14mu}{group}}\end{matrix}}{{parasitaemia}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{group}} \times 100\;\%}$

Method: P. flaciparum FCC1/HN line was cultured in vitro as described byTrager and Jansen. The culture medium was refreshed every 24 hrs andfresh red blood cells were added to culture every four day. To preparesynchronous parasites for the inhibition assay, the infectederythrocytes were mixed with 5% sorbitol and incubated at RT for 30minutes. The treated parasite was further cultured at 30° C. for 24hours. Thus most parasites developed into schizonts. The initial culturewas prepared to have 2% haematocrit and 0.5% parasitaemia. 200 ul of theculture was added to each well containing different concentration oftest sera. After 24 or 72 hours, thin smears were made and stained withGiemsa's solution to determine parasitaemia under the microscope.

Preparation of anit-sera: The rabbit blood was taken by cardiac punctureand put into sterile tubes to wait for coagulation. The anti-sera werecollected from coagulation by centrifugation. sera was heat-inactivatedat 56° C. 30 min and sterilized by filtration before using forinhibition assays.

Deletion of IgG from immune sera: The IgG in immune sera was eliminatedby Protein A column according to the manufactory instruction. This stepwas repeated until no IgG was observed in the flowthrough detected bySDS-PAGE. Moreover, bounded IgG was eluted from the column.

The results of in vitro inhibition assay showed that immune sera fromrabbits immunized with PfCP-2 antigen formulated either with Freund'sadjuvant or ISA 720 adjuvant inhibited the growth of more than 98%parasites after diluted at 6.7 times. The inhibition was dependent onpresence of the specific antibodies and the conformation of PfCP-2.(FIGS. 13–16)

Result:

(1) The immune sera obtained from rabbits immunized with PfCP-2formulated with Freund's adjuvant inhibited 96.3% parasite growth whilesera from Freund's adjuvant control had no significant effect onparasite growth. (IR: 6.41%) (FIG. 13)

(2) The immune sera obtained from rabbits immunized with PfCP-2formulated with ISA720 adjuvant inhibited 98.32% parasite growth whilesera from ISA720 adjuvant control had no significant effect on parasitegrowth. (IR: 0.69%) (FIG. 14)

(3) The immune sera obtained from rabbits immunized with denaturedPfCP-2 protein by DTT as well as 8M urea and alkylated, formulated withISA 720 adjuvant did not inhibit parasite growth at all. (IR: 0%) (FIG.15)

(4) Immune sera removed of all IgG by protein A column was tested forinhibition of parasite growth in vitro. The result showed thatIgG-eliminating sera from rabbits immunized with the antigen formulatedwith both ISA720 and Freund's adjuvant had no effective on the parasitegrowth with inhibition rates of 0% and 12.7%, respectively (FIG. 16).

The study on the Growth Inhibition Assay indicated: (1) The immune serainduced by PfCP-2 almost completely inhibited the parasite growth invitro(IR: >98%) when diluted at 6.7 times and the inhibition wasdependent on presence of IgG basing on the fact that the sera removed oftotal IgG was not shown to inhibit parasite growth. (2) AlthoughFreund's adjuvant is a powerful adjuvant, it can be used only inanimals. However, ISA 720 adjuvant (produced by French SEPPIC Co.) hasbeen used in clinical trials. Our results showed that the antibody levelinduced with ISA 720 was comparable to that with Freund's adjuvant.Thus, ISA720 adjuvant could be an appropriate adjuvant for PfCP-2vaccine candidate in human use. (3) Induction of inhibitory antibodiesto PfCP-2 were dependent on its conformation because the denatured agentdid not induce any inhibitory antibodies. Therefore, it is essential toproduce PfCP-2 recombinant protein resembling closely to its nativeconformation.

EXAMPLE 7 Pfcp-2 Combined Vaccine

In this example, the combined vaccine of Pfcsp-2 and other Plasmodiumcircumsporozoite protein or its immunogenetic fragment were prepared.Pfcsp^(RC) was specifically used which had a part of the sequence ofPlamodium falciparum circumsporozoite protein, comprising 15 NANPrepeating sequences and the whole C-terminus sequence. The whole gene ofPfcsp^(RC) was synthesized by using the codon preferred for PichiaHansen, highly secreted and expressed in Pichia Hansen, and was isolatedand purified to get Pfcsp^(RC).

The immunity produced by Pfcsp-2 is directed against early phasePlasmodium, and the immunity produced by Pfcsp^(RC) is directed againstlate phase Plasmodium.

The result of co-immunization of rabbit with purified Pfcsp^(RC) andPfcp-2 showed that, after 4 tims immunization, the antibody titer of thegroup immunized with Pfcp-2 alone was 1/1,604,000, and the antibodytiter of the group immunized with Pfcs^(RC) was 1/136,000. However, theco-immunized group produced antibodies directed at both antigens withremarkable increase in titers. The antibody titer against Pfcp-2 andPfcsp^(RC) were 1/2,859,000 and 1/443,000, respectively. This resultsuggests that the combined immunization of two antigens can not onlyproduce antibodies against Plasmodium in different phases, but alsoimprove the immunogenicity of the two antigens.

The antibody assay for rabbits combination immunized with Pfcp-2 andPfcsp^(RC) (×10⁴) coating blood collecting immunogen antigen time Pfcp-2Pfcsp^(RC) Pfcp-2+Pfcso^(RC) Pfcp-2 after 2^(nd) 39.52 — 36.02immunization after 3^(rd) 108.60 — 107.74 immunization after 4^(th)164.40 — 285.90 immunization Pfcsp^(RC) after 2^(nd) — 4.41 5.35immunization after 3^(rd) — 4.41 9.56 immunization after 4^(th) — 13.6044.30 immunization note: 1. The values in the table were geometricalmeans for 3 rabbits. 2. Dose of immunization: The dose of Pfcp-2 andPfcsp^(RC) alone were 200 μg, respectively. The dose of combinationimmunized group is 200 μg Pfcp-2 and 200 μg Pfcsp^(RC). 3. The adjuvantused in the immunization experiment is ISA720, i.m. 4. The values in thetable were antibody titers assayed by ELISA.

All the documents cited herein are incorporated into the invention asreference, as if each of them is individually incorporated. Further, itwould be appreciated that, in the above teaching of invention, theskilled in the art could make certain changes or modifications to theinvention, and these equivalents would still be within the scope of theinvention defined by the appended claims of the application.

1. A fusion protein comprising: an amino acid sequence of Plasmodiumapical membrane antigen-1 (AMA-1), an amino acid sequence of Plasmodiummerozoite surface protein 1 (MSP1), and a hinge between the amino acidsequence of the apical membrane antigen-1 and the amino acid sequence ofthe merozoite surface protein 1, wherein the amino acid sequence ofAMA-1 is selected from the group consisting of the amino acid sequenceof natural full-length AMA-1, the amino acid sequence of the wholeectodomain of AMA-1, the amino acid sequence of domain III of AMA-1, andthe amino acid sequence of domain I-III of AMA-1; and the amino acidsequence of MSP1 is selected from the group consisting of the amino acidsequence of natural full-length MSP1 and the amino acid sequence of MSP119KD C-terminal; the hinge comprising an amino acid sequence selectedfrom the group consisting of: (a) an amino acid sequence containing 6amino acids comprising hydrophobic amino acids Gly and Pro; (b) an aminoacid sequence encoded by multiple cloning sites; and (c) a combinationof (a) and (b).
 2. The fusion protein according to claim 1 wherein thehinge contains: an amino acid sequence containing 6 amino acids made ofhydrophobic amino acids Gly and Pro.
 3. The fusion protein according toclaim 1 comprising an amino acid sequence set forth in SEQ ID NO: 1, 2or
 3. 4. An isolated DNA molecule encoding the fusion protein ofclaim
 1. 5. A vector comprising the DNA molecule of claim
 4. 6. A hostcell comprising the vector of claim
 5. 7. The host cell according toclaim 6, wherein the cell is a yeast Pichia pastoris deposited in CCTCCunder accession Number of CCTCC NO: M200026.
 8. A method of preparingthe fusion protein of claim 1 comprising the steps of: growing the hostcell of claim 6 under a condition appropriate for the expression,thereby expressing the fusion protein, and isolating the fusion protein.9. A method for producing a polyclonal antibody, which inhibits thegrowth of P. falciparum in vitro, comprising the following steps: (i)administering the fusion protein of claim 1 to an animal, therebyinducing the generation of a polyclonal antibody; and (ii) isolating thepolyclonal antibody.
 10. The method of claim 9 wherein the fusionprotein comprises an amino acid sequence set forth in SEQ ID NO: 1, 2 or3.